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La,25-Dihydroxyvitamin D3 Regulates the Expression of Carbonic

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La,25-Dihydroxyvitamin D3 Regulates the Expression of Carbonic Proc. Natl. Acad. Sci. USA Vol. 87, pp. 6470-6474, August 1990 Cell Biology la,25-Dihydroxyvitamin D3 regulates the expression of carbonic anhydrase II in nonerythroid avian bone marrow cells (myelomonocytes/osteoclasts/macrophages/bone resorption/acid-base regulation) AGNES BILLECOCQ*t, JANET RETTIG EMANUEL*, ROBERT LEVENSON*, AND ROLAND BARON*t§ Departments of *Cell Biology and tOrthopedics, Yale University School of Medicine, P.O. Box 3333, New Haven, CT 06510 Communicated by Robert W. Berliner, May 8, 1990 ABSTRACT la,25-Dihydroxyvitamin D3 [1,25(OH)2D3], such as the kidney tubule intercalated cell (26), the gastric the active metabolite of the steroid hormone vitamin D, is a mucosa oxyntic cell (27), and the osteoclast (21). In humans, potent regulator of macrophage and osteoclast differentiation. defective CAII is associated with cerebral calcification, tu- The mature osteoclast, unlike the circulating monocyte or the bular acidosis, and osteopetrosis (28), providing genetic tissue macrophage, expresses high levels ofcarbonic anhydrase evidence for CAII involvement in bone resorption. An ana- H (CAH). This enzyme generates protons and bicarbonate logue of the human disease has been produced by chemical from water and carbon dioxide and is involved in bone resorp- mutagenesis in the mouse, where CAII deficiency is associ- tion and acid-base regulation. To test whether 1,25(OH)2D3 ated with renal tubular acidosis and vascular calcification but could induce the differentiation of myelomonocytic precursors no major bone defects (29). toward osteoclasts rather than macrophages, we analyzed its We have investigated whether CAII expression is regu- effects on the expression of CAll in bone marrow cultures lated in bone marrow cultures by 1,25(OH)2D3. We find that containing precursors common to both cell types. The expres- the levels of expression of CAII mRNA and protein are sion ofCAU was markedly increased by 1,25(OH)2D3 in a dose- markedly increased by 1,25(OH)2D3 during the in vitro dif- and time-dependent manner. In bone marrow, this increase ferentiation ofnormal bone marrow precursors. This was also occurred at the mRNA and protein levels and was detectable as observed in a transformed myelomonocytic cell line (BM2), early as 24 hr after stimulation. 1,25(OH)2D3 was also found to thereby confirming a recent report in the transformed human induce CAH expression in a transformed myelomonocytic monocytic cell line HL60 (30). These results suggest that avian cell line. These results suggest that 1,25(OH)2D3 regulates 1,25(OH)2D3 acts directly on myelomonocytic precursor cells the level at which myelomonocytic precursors express CAU, an to induce expression of CAII, a protein that is present at high enzyme that is involved in the function ofthe mature osteoclast. levels in the fully differentiated osteoclast. la,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is the active me- MATERIAL AND METHODS tabolite ofthe steroid hormone vitamin D (1). This metabolite Cell Preparations. Bone marrow cells were isolated from regulates the differentiation of osteoclasts and macrophages, the tibias and femurs of White Leghorn chickens (SPAFAS, two cell types that are thought to be derived from a common Norwich, CT) fed a calcium-deficient diet (Purina) for 1-2 myelomonocytic precursor (2-6). 1,25(OH)2D3 induces an weeks after hatching. Cells passed through a 10-,um nylon increase in bone resorption in vivo (1, 7) and in organ cultures filter (Small Parts) to remove mature multinucleated osteo- (8, 9) but has no effect on the resorptive activity of isolated clasts were fractionated on Ficoll-Paque (Pharmacia) and osteoclasts (10, 11). The increase in bone resorption is plated for 16 hr at 5 x 106 cells per ml in alpha minimum believed to be mediated primarily by means of an effect upon essential medium (Sigma) containing heat-inactivated chick- the proliferation and/or differentiation of osteoclast precur- en (2%) and fetal calf (8%) sera (Sigma), 50 units of penicillin sors within the hematopoietic bone marrow (12-15), leading per ml, and 50 ,ug of streptomycin per ml. To deplete these to an increase in the number of mature osteoclasts (7, 9). This preparations of the most mature stromal and myelomono- view is supported by in vitro experiments with normal bone cytic cells (31), nonadherent cells were recovered after a marrow cells or transformed cell lines. First, 1,25(OH)2D3 16-hr culture and replated at 106 cells per ml for up to 6 days. induces the differentiation and maturation of myelomono- The cells were plated in 96-well plates (105 cells per well) for cytic cell lines toward the mononuclear phagocyte rather than ELISA, on glass coverslips in 24-well plates for immunocy- the granulocyte lineage (3, 12, 15-18). Further, 1,25(OH)2D3 tochemistry (5 x 105 cells per well), and in 150-cm2 flasks (2.5 stimulates the formation of multinucleated osteoclast-like x i05 cells per cm2) for immunochemistry or RNA extrac- cells in long-term bone marrow cultures (13, 14, 19, 20) and tion. Cells were grown in the absence or presence of 10-11 to increases the proportion ofthe cells that can resorb bone (14). 10-7 M 1,25(OH)2D3 (provided by Milan Uskokovic, Hoff- Taken together, these results suggest that this steroid hor- man-LaRoche). mone may specifically induce the differentiation of my- ELISA. After 1-6 days of culture, cells were fixed in 3.7% elomonocytic precursors toward the macrophage and/or formaldehyde, treated with 0.01% H202, and preincubated osteoclast lineage. for 1 hr in phosphate-buffered saline (PBS)/3% bovine serum One of the genes that is expressed at high levels in the albumin. Cells were incubated for 2 hr with antibodies (Abs) osteoclast (21-23), but not in peripheral monocytes or mature diluted in PBS/3% bovine serum albumin with saponin macrophages (24), is the gene encoding carbonic anhydrase (0.05%) to permeabilize the cells. After washing, cells were II (CAII). CAII is an enzyme that reversibly generates bicarbonate and protons from carbon dioxide and water (25). Abbreviations: 1,25(OH)2D3, la,25-dihydroxyvitamin D3; CAII, car- This enzyme plays an essential role in acid-secreting cells bonic anhydrase II; Ab, antibody; mAb, monoclonal Ab; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; AMV, avian myeloblastosis virus. The publication costs of this article were defrayed in part by page charge tPresent address: Department of Experimental Pathophysiology, payment. This article must therefore be hereby marked "advertisement" Pasteur Institute, Paris, France. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 6470 Cell Biology: Billecocq et al. Proc. Natl. Acad. Sci. USA 87 (1990) 6471 incubated with peroxidase-labeled goat anti-mouse IgG Ab RPMI 1640 containing 5% heat-inactivated calf serum, 5% (Cappel) diluted 1:1000 in PBS/3% bovine serum albumin, heat-inactivated chicken serum, 50 units of penicillin per ml, washed, and allowed to react with 2 mg of o-phenylenedi- and 50 pug of streptomycin per ml. The cells were cultured for amine per ml (Sigma) and 0.03%1H202. The reaction was 6 days in the absence or presence of 10 Ag of LPS per ml and stopped after 15 min by the addition of H2SO4. Optical 0.25 pug of PMA per ml. 1,25(OH)2D3 (10-8 M) was added to densities (ODs) were read in duplicate on an ELISA reading some cultures at day 0. Cells were processed for immunocy- system (EAR400, Labinstruments) at 492/620 nm. Two tochemistry with the mAb to CANl as described above. monoclonal antibodies (mAbs) were used: mouse anti- chicken CAII as culture supernatant [7C6-1 (32) diluted 1:20] RESULTS and anti-LEP100 as ascites fluid [(33) diluted 1:1000]. The We analyzed the expression of CAII in bone marrow cultures background OD, measured without primary Ab or with an immunoblots with an antiserum that reacts spe- irrelevant Ab [anti-dinitrophenyl (gift of Richard Anderson, by probing University of Texas, Dallas) diluted 1:200], was subtracted cifically with CAII (36). A marked enhancement in the level from the ODs measured with the specific Abs. In three of the immunoreactive 29-kDa CAII band was observed experiments the protein contents were measured simultane- following 6 days of incubation in the presence of 10-9 M ously (34) after lysis in 0.1% SDS. 1,25(OH)2D3 (Fig. 1). In contrast, cells grown in the absence Western Blots. Cells were washed with PBS, pelleted, and of 1,25(OH)2D3 contained either undetectable (Fig. 1A) or lysed at 40C in lysis buffer [150 mM NaCl containing 1% very low (Fig. 1B) levels of CAI. Quantitation of the Triton X-100, 0.2% SDS, and protease inhibitors (1 mM immunoreactive bands indicated that in response to phenylmethylsulfonyl fluoride, 5 mM EDTA, 10 pug of leu- 1,25(OH)2D3 the levels of CAII increased from 4- to >14-fold. peptin per ml, 1 mM dithiothreitol, and 2 mM benzamidine) To determine whether this effect occurred in a dose-depen- in 50mM Tris buffer at pH 7.4]. Proteins were measured (34), dent manner, CAII levels were determined by ELISA, using electrophoresed through SDS/PAGE (10%), transferred to a CAII-specific mAb (32), in cultures treated with various nitrocellulose (35), and incubated with a rabbit anti-chicken amounts of vitamin D3. Expression of CAII was compared to CAII polyclonal Ab (36) or nonimmune serum diluted 1:2000 that of LEP100, a lysosomal membrane protein (33) that was in Tris-buffered saline. Nitrocellulose sheets were incubated found to vary proportionally to protein content and not to for 2 hr with 0.5 ,Ci of "25I-labeled protein A (Amersham; 1 levels of 1,25(OH)2D3 (data not shown).
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